Hepatitis C virus (HCV) is the major etiological agent of post-transfusion and community-acquired non-A non-B hepatitis worldwide. It is estimated that over 150 million people worldwide are infected by the virus. A high percentage of carriers become chronically infected and many progress to chronic liver disease, so-called chronic hepatitis C. This group is in turn at high risk for serious liver disease such as liver cirrhosis, hepatocellular carcinoma and terminal liver disease leading to death.
The mechanism by which HCV establishes viral persistence and causes a high rate of chronic liver disease has not been thoroughly elucidated. It is not known how HCV interacts with and evades the host immune system. In addition, the roles of cellular and humoral immune responses in protection against HCV infection and disease have yet to be established. Immunoglobulins have been reported for prophylaxis of transfusion-associated viral hepatitis, however, the Center for Disease Control does not presently recommend immunoglobulins treatment for this purpose. The lack of an effective protective immune response is hampering the development of a vaccine or adequate post-exposure prophylaxis measures, so in the near-term, hopes are firmly pinned on antiviral interventions.
Various clinical studies have been conducted with the goal of identifying pharmaceutical agents capable of effectively treating HCV infection in patients afflicted with chronic hepatitis C. These studies have involved the use of interferon-alpha, alone and in combination with other antiviral agents. Such studies have shown that a substantial number of the participants do not respond to these therapies, and of those that do respond favorably, a large proportion were found to relapse after termination of treatment.
Until recently, interferon (IFN) was the only available therapy of proven benefit approved in the clinic for patients with chronic hepatitis C. However the sustained response rate is low, and interferon treatment also induces severe side-effects (i.e. retinopathy, thyroiditis, acute pancreatitis, depression) that diminish the quality of life of treated patients. Recently, interferon in combination with ribavirin has been approved for patients non-responsive to IFN alone. However, the side effects caused by IFN are not alleviated with this combination therapy.
Therefore, a need exists for the development of effective antiviral agents for treatment of HCV infection that overcomes the limitations of existing pharmaceutical therapies.
HCV is an enveloped positive strand RNA virus in the Flaviviridae family. The single strand HCV RNA genome is approximately 9500 nucleotides in length and has a single open reading frame (ORF) encoding a single large polyprotein of about 3000 amino acids. In infected cells, this polyprotein is cleaved at multiple sites by cellular and viral proteases to produce the structural and non-structural (NS) proteins. In the case of HCV, the generation of mature nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) is effected by two viral proteases. The first one, as yet poorly characterized, cleaves at the NS2-NS3 junction; the second one is a serine protease contained within the N-terminal region of NS3 (henceforth referred to as NS3 protease) and mediates all the subsequent cleavages downstream of NS3, both in cis, at the NS3-NS4A cleavage site, and in trans, for the remaining NS4A-NS4B, NS4B-NS5A, NS5A-NS5B sites. The NS4A protein appears to serve multiple functions, acting as a cofactor for the NS3 protease and possibly assisting in the membrane localization of NS3 and other viral replicase components. The complex formation of the NS3 protein with NS4A seems necessary to the processing events, enhancing the proteolytic efficiency at all of the sites. The NS3 protein also exhibits nucleoside triphosphatase and RNA helicase activities. NS5B is a RNA-dependent RNA polymerase that is involved in the replication of HCV. A general strategy for the development of antiviral agents is to inactivate virally encoded enzymes that are essential for the replication of the virus. In this vein, patent application WO 97/06804 describes the (-) enantiomer of the nucleoside analogue cytosine-1,3-oxathiolane (also known as 3TC) as active against HCV. This compound, although reported as safe in previous clinical trials against HIV and HBV, has yet to be clinically proven active against HCV and its mechanism of action against the virus has yet to be reported.
Intense efforts to discover compounds which inhibit the NS3 protease or RNA helicase of HCV have led to the following disclosures:
U.S. Pat. No. 5,633,388 describes heterocyclic-substituted carboxamides and analogues as being active against HCV. These compounds are directed against the helicase activity of the NS3 protein of the virus but clinical tests have not yet been reported. PA1 A phenanthrenequinone has been reported by Chu et al., (Tet. Lett., (1996), 7229-7232) to have activity against the HCV NS3 protease in vitro. No further development on this compound has been reported. PA1 A paper presented at the Ninth International Conference on Antiviral Research, Urabandai, Fukyshima, Japan (1996) (Antiviral Research, (1996), 30, 1, A23 (abstract 19)) reports thiazolidine derivatives to be inhibitory to the HCV protease. PA1 WO 98/17679 from Vertex Pharmaceuticals Inc. discloses inhibitors of serine protease, particularly, Hepatitis C virus NS3 protease. These inhibitors are peptide analogues based on the NS5A/5B natural substrate. Although several tripeptides are disclosed, all of these peptide analogues contain C-terminal activated carbonyl function as an essential feature. These analogues were also reported to be active against other serine protease and are therefore not specific for HCV NS3 protease. PA1 Hoffman LaRoche has also reported hexapeptides that are proteinase inhibitors useful as antiviral agents for the treatment of HCV infection. These peptides contain an aldehyde or a boronic acid at the C-terminus. PA1 Steinkuhler et al. and Ingallinella et al. have published on NS4A-4B product inhibition (Biochemistry (1998), 37, 8899-8905 and 8906-8914). However, the peptides and peptide analogues presented do not include nor do they lead to the design of the peptides of the present invention. PA1 B is H, a C.sub.6 or C.sub.10 aryl, C.sub.7-16 aralkyl; Het or (lower alkyl)-Het, all of which optionally substituted with C.sub.1-6 alkyl; C.sub.1-6 alkoxy; C.sub.1-6 alkanoyl; hydroxy; hydroxyalkyl; halo; haloalkyl; nitro; cyano; cyanoalkyl; amino optionally substituted with C.sub.1-6 alkyl; amido; or (lower alkyl)amide; PA1 or B is an acyl derivative of formula R.sub.4 --C(O)--; a carboxyl derivative of formula R.sub.4 --O--C(O)--; an amide derivative of formula R.sub.4 --N(R.sub.5)--C(O)--; a thioamide derivative of formula R.sub.4 --N(R.sub.5)--C(S)--; or a sulfonyl derivative of formula R.sub.4 --SO.sub.2 wherein PA1 Y is H or C.sub.1-6 alkyl; PA1 R.sup.3 is C.sub.1-8 alkyl, C.sub.3-7 cycloalkyl, or C.sub.4-10 alkylcycloalkyl, all optionally substituted with hydroxy, C.sub.1-6 alkoxy, C.sub.1-6 thioalkyl, amido, (lower alkyl)amido, C.sub.6 or C.sub.10 aryl, or C.sub.7-16 aralkyl; PA1 R.sub.2 is CH.sub.2 -R.sub.20, NH-R.sub.20, O-R.sub.20 or S-R.sub.20, wherein R.sub.20 is a saturated or unsaturated C.sub.3-7 cycloalkyl or C.sub.4-10 (alkylcycloalkyl), all of which being optionally mono-, di- or tri-substituted with R.sub.21, or R.sub.20 is a C.sub.6 or C.sub.10 aryl or C.sub.7-14 aralkyl, all optionally mono-, di- or tri-substituted with R.sub.21, or R.sub.20 is Het or (lower alkyl)-Het, both optionally mono-, di- or tri-substituted with R.sub.21, PA1 R.sup.1 is H, C.sub.1-6 alkyl, C.sub.3-7 cycloalkyl, C.sub.2-6 alkenyl, or C.sub.2-6 alkynyl, all optionally substituted with halogen; PA1 in which the R moiety of the ester is selected from alkyl (e.g. methyl, ethyl, n-propyl, t-butyl, n-butyl); alkoxyalkyl (e.g. methoxymethyl); alkoxyacyl (e.g. acetoxymethyl); aralkyl (e.g. benzyl); aryloxyalkyl (e.g. phenoxymethyl); aryl (e.g. phenyl), optionally substituted with halogen, C.sub.1-4 alkyl or C.sub.1-4 alkoxy. Other suitable prodrug esters can be found in Design of prodrugs, Bundgaard, H. Ed. Elsevier (1985) incorporated herewith by reference. Such pharrhaceutically acceptable esters are usually hydrolyzed in vivo when injected in a mammal and transformed into the acid form of the compound of formula I. PA1 Preferably, B is a C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C.sub.1-6 alkyl; or PA1 B is preferably Het or (lower alkyl)-Het, all optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C.sub.1-6 alkyl. PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, hydroxy or C.sub.1-6 alkoxy, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionally substituted with C.sub.1-6 alkyl; PA1 (v) Het or (lower alkyl)-Het, both optionally substituted with C.sub.1-6 alkyl, hydroxy, amino optionally substituted with C.sub.1-6 alkyl, amido, (lower alkyl)amide, or amino optionally substituted with C.sub.1-6 alkyl. PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, amido or (lower alkyl)amide; PA1 (ii) C.sub.3-7 cycloalkyl, C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, amido or (lower alkyl)amide; PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl optionally substituted with C.sub.1-6 alkyl, hydroxy, amido, (lower alkyl)amido, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; or PA1 (v) Het or (lower alkyl)-Het, both optionally substituted with C.sub.1-6 alkyl, hydroxy, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, amido or (lower alkyl)amido. PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy, amido, (lower alkyl)amido, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amido, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (iii) amino optionally mono- or di-substituted with C.sub.1-3 alkyl; PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, amido, (lower alkyl)amide, or amino optionally substituted with C.sub.1-6 alkyl; or PA1 (v) Het or (lower alkyl)-Het, both optionally substituted with C.sub.1-6 alkyl, hydroxy, amino optionally substituted with C.sub.1-6 alkyl, amido or (lower alkyl)amide; and PA1 R.sub.5 is preferably H or methyl. PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl or C.sub.1-6 alkoxy; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amino or amido. PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, hydroxy or C.sub.1-6 alkoxy; or PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, both optionally substituted with hydroxy, carboxyl, (C.sub.1-6 alkoxy)carbonyl, such that B is, for example: ##STR16## PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, such that B is for example: ##STR17## PA1 (v) Het optionally substituted with C.sub.1-6 alkyl, hydroxy, amido or amino, such that B is for example: ##STR18## PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy or amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl, C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, such that B is for example: ##STR19## PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, amino optionally substituted with C.sub.1-6 alkyl; or PA1 (v) Het or (lower alkyl)-Het, both optionally substituted with C.sub.1-6 alkyl, hydroxy, amido, or amino optionally mono-substituted with C.sub.1-6 alkyl, such that B is for example: ##STR20## PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; and PA1 (iii) amino optionally mono- or di-substituted with C.sub.1-3 alkyl, such that B is for example: ##STR22## PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, hydroxy, amino or amido optionally substituted with C.sub.1-6 alkyl; or PA1 (v) Het optionally substituted with C.sub.1-6 alkyl, hydroxy, amino or amido, such that B is for example: ##STR23## PA1 R.sub.4 is (i) C.sub.1-10 alkyl; or (ii) C.sub.3-7 cycloalkyl, such that B is for example: ##STR24## PA1 (i) C.sub.1-10 alkyl optionally substituted with carboxyl, C.sub.1-6 alkanoyl, hydroxy, C.sub.1-6 alkoxy amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; PA1 (ii) C.sub.3-7 cycloalkyl or C.sub.4-10 alkylcycloalkyl, all optionally substituted with carboxyl, (C.sub.1-6 alkoxy)carbonyl, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl; ##STR25## PA1 (iv) C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl optionally substituted with C.sub.1-6 alkyl, hydroxy, amino or amido, such that B is for example: ##STR26## PA1 Preferably, R.sub.21 is C.sub.1-6 alkyl; C.sub.1-6 alkoxy; lower thioalkyl; amino or amido optionally mono-or di-substituted with C.sub.1-6 alkyl, C.sub.6 or C.sub.10 aryl, C.sub.7-16 aralkyl, Het or (lower alkyl)-Het; NO.sub.2 ; OH; halo; trifluoromethyl; catboxyl; C.sub.6 or C.sub.10 aryl, C.sub.7-16 aralkyl, or Het, said aryl, aralkyl or Het being optionally substituted with R.sub.22. More preferably, R.sub.21 is C.sub.1-6 alkyl; C.sub.1-6 alkoxy; amino; di(lower alkyl)amino; (lower alkyl)amide; C.sub.6 or C.sub.10 aryl, or Het, said aryl or Het being optionally substituted with R.sub.22. PA1 lower thioalkyl such as ##STR34## PA1 halo such as chloro; PA1 amino optionally mono-substituted with C.sub.1-6 alkyl; or C.sub.6 or C.sub.10 aryl, such that R.sub.21A is for example: dimethylamino, Ph--N(Me)--; PA1 unsubstituted C.sub.6 or C.sub.10 aryl, C.sub.7-16 aralkyl, such as for example phenyl or ##STR35## PA1 wherein R.sub.22A is preferably C.sub.1-6 alkyl (such as methyl); C.sub.1-6 alkoxy (such as methoxy); or halo (such as chloro); R.sub.22B is preferably C.sub.1-6 alkyl, amino optionally mono-substituted with C.sub.1-6 alkyl, amido; or (lower alkyl)amide; and R.sub.21B is preferably C.sub.1-6 alkyl, C.sub.1-6 alkoxy, amino, di(lower alkyl)amino, (lower alkyl)amide, NO.sub.2, OH, halo, trifluoromethyl, or carboxyl. More preferably, R.sub.21B is C.sub.1-6 alkoxy, or di(lower alkyl)amino. Most preferably, R.sub.21B is methoxy. PA1 wherein C.sub.1 and C.sub.2 each represent an asymmetric carbon atom at positions 1 and 2 of the cyclopropyl ring. Not withstanding other possible asymmetric centers at other segments of the compounds of formula I, the presence of these two asymmetric centers means that the compounds of formula I can exist as racemic mixtures of diastereoisomers. As illustrated in the examples hereinafter, the racemic mixtures can be prepared and thereafter separated into individual optical isomers, or these optical isomers can be prepared by chiral synthesis. PA1 B is a C.sub.6 or C.sub.10 aryl or C.sub.7-16 aralkyl, all optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C.sub.1-6 alkyl; or Het or (lower alkyl)-Het, all optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amido, or amino optionally substituted with C.sub.1-6 alkyl, or PA1 B is R.sub.4 --SO.sub.2 wherein R.sub.4 is preferably amido; (lower alkyl)amide; C.sub.6 or C.sub.10 aryl, C.sub.7-14 aralkyl or Het, all optionally substituted with C.sub.1-6 alkyl, or PA1 B is an acyl derivative of formula R.sub.4 --C(O)-- wherein R.sub.4 is PA1 B is an carboxyl of formula R.sub.4 --O--C(O)--, wherein R.sub.4 is PA1 B is an amide of formula R.sub.4 --N(R.sub.5)--C(O)-- wherein R.sub.4 is PA1 R.sub.5 is preferably H or methyl, or PA1 B is thioamide of formula R.sub.4 --NH--C(S)--; wherein R.sub.4 is PA1 Y is H or methyl; PA1 R.sup.3 is C.sub.1-8 alkyl, C.sub.3-7 cycloalkyl, or C.sub.4-10 alkylcycloalkyl, all optionally substituted with hydroxy, C.sub.1-6 alkoxy, C.sub.1-6 thioalkyl, acetamido, C.sub.6 or C.sub.10 aryl, or C.sub.7-16 aralkyl; PA1 R.sup.2 is S--R.sub.20 or O--R.sub.20 wherein R.sub.20 is preferably a C.sub.6 or C.sub.10 aryl, C.sub.7-16 aralkyl, Het or --CH.sub.2 -Het, all optionally mono-, di- or tri-substituted with R.sub.21, wherein PA1 R.sub.2 is selected from the group consisting of: ##STR43## PA1 or R.sub.2 is 1-naphthylmethoxy; 2-naphthylmethoxy; benzyloxy, 1-naphthyloxy; 2-naphthyloxy; or quinolinoxy unsubstituted, mono- or di-substituted with R.sub.21 as defined above; PA1 the P1 segment is a cyclobutyl or cyclopropyl ring, both optionally substituted with R.sup.1, wherein R.sup.1 is H, C.sub.1-3 alkyl, C.sub.3-5 cycloalkyl, or C.sub.2-4 alkenyl optionally substituted with halo, and said R.sup.1 at carbon 2 is orientated syn to the carbonyl at position 1, represented by the radical: ##STR44## PA1 B is a C.sub.6 or C.sub.10 aryl optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkanoyl, hydroxy, hydroxyalkyl, halo, haloalkyl, nitro, cyano, cyanoalkyl, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; or B is Het optionally substituted with C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 alkanoyl, hydroxy, halo, amido, (lower alkyl)amide, or amino optionally mono- or di-substituted with C.sub.1-6 alkyl; or B is R.sub.4 --SO.sub.2 wherein R.sub.4 is C.sub.6 or C.sub.10 aryl, a C.sub.7-14 aralkyl or Het all optionally substituted with C.sub.1-6 alkyl; amido, (lower alkyl)amide; or B is an acyl derivative of formula R.sub.4 --C(O)-- wherein R.sub.4 is PA1 or B is a carboxyl of formula R.sub.4 --O--C(O)--, wherein R.sub.4 is PA1 or B is an amide of formula R.sub.4 --N(R.sub.5)--C(O)-- wherein R.sub.4 is PA1 B is a thioamide of formula R.sub.4 --NH--C(S)--; wherein R.sub.4 is: PA1 B is an amide of formula R.sub.4 --NH--C(O)-- wherein R.sub.4 is PA1 Y is H; PA1 R.sup.3 is the side chain of tert-butylglycine (Tbg), Ile, Val, Chg or; ##STR45## PA1 R.sup.2 is 1-naphtylmethoxy; or quinolinoxy unsubstituted, mono- or di-substituted with R.sub.21 as defined above, or PA1 R.sup.2 is: ##STR46## PA1 P1 is a cyclopropyl ring wherein carbon 1 has the R configuration, ##STR47## PA1 and R.sup.1 is ethyl, vinyl, cyclopropyl, 1 or 2-bromoethyl or 1 or 2-bromovinyl. PA1 B is tert-butoxycarbonyl (Boc) or PA1 R.sup.3 is the side chain of Tbg, Chg or Val; PA1 R.sup.2 : ##STR49## PA1 and P1 is: ##STR50## PA1 Generally, peptides are elongated by deprotecting the .alpha.-amino group of the N-terminal residue and coupling the unprotected carboxyl group of the next suitably N-protected amino acid through a peptide linkage using the methods described. This deprotection and coupling procedure is repeated until the desired sequence is obtained. This coupling can be performed with the constituent amino acids in stepwise fashion, as depicted in Scheme I, or by solid phase peptide synthesis according to the method originally described in Merrifield, J. Am. Chem. Soc., (1963), 85, 2149-2154, the disclosure of which is hereby incorporated by reference. Coupling between two amino acids, an amino acid and a peptide, or two peptide fragments can be carried out using standard coupling procedures such as the azide method, mixed carbonic-carboxylic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimide) method, active ester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method, Woodward reagent K-method, carbonyidiimidazole method, phosphorus reagents or oxidation-reduction methods. Some of these methods (especially the carbodiimide method) can be enhanced by adding 1-hydroxybenzotriazole. These coupling reactions can be performed in either solution (liquid phase) or solid phase. PA1 b) Copper catalyzed couplings according to Ma et al. (J. Am. Chem. Soc. 1998, 120, 12459-12467): ##STR53## PA1 c) Nucleophilic displacement of a triflate by an aniline: ##STR54## PA1 a) The Fmoc-thiocyanate prepared according to Kearney et al., 1998, J. Org. Chem, 63, 196, was reacted with a protected P3 residue or the whole peptide or a peptide segment to provide the thiourea. PA1 b) The thiourea derivative is reacted with an appropriate bromoketone to provide the corresponding thiazole derivative. PA1 a) Cyclobutanol is treated with phosgene to furnish the corresponding chloroformate. PA1 b) The chloroformate is treated with the desired NH.sub.2 -tripeptide in the presence of a base such as triethylamine to afford the cyclobutylcarbamate. PA1 R.sub.22 & R.sub.21B =alkyl, OH, SH, halo, NH.sub.2, NO.sub.2. PA1 (wherein R.sup.2 is attached to the ring via a carbon atom) (with the stereochemistry as shown): ##STR59## PA1 is done as shown in Scheme IV according to the procedures described by J. Ezquerra et al. (Tetrahedron, (1993), 38, 8665-8678) and C. Pedregal et al. (Tetrahedron Lett., (1994), 35, 2053-2056). ##STR60## PA1 1) When R.sup.20 is aryl, aralkyl, Het or (lower alkyl)-Het, the process can be carried out according to the procedure described by E. M. Smith et al. (J. Med. Chem. (1988), 31, 875-885). Briefly, commercially available Boc-4(R)-hydroxyproline is treated with a base such as sodium hydride or potassium tert-butoxide and the resulting alkoxide reacted with halo-R.sup.20 (Br--R.sup.20, I--R.sup.20, etc.) to give the desired compounds. Specific embodiments of this process are presented in Examples 4, 5 and 7. PA1 2) Alternatively, when R.sup.20 is aryl or Het, the compounds can also be prepared via a Mitsunobu reaction (Mitsunobu (1981), Synthesis, January, 1-28; Rano et al., (1995), Tet. Lett. 36(22), 3779-3792; Krchnak et al., (1995), Tet. Lett. 36(5), 62193-6196; Richter et al., (1994), Tet. Lett. 35(27), 4705-4706). Briefly, commercially available Boc-4(S)-hydroxyproline methyl ester is treated with the appropriate aryl alcohol or thiol in the presence of triphenylphosphine and diethylazodicarboxylate (DEAD) and the resulting ester is hydrolyzed to the acid. Specific embodiments of this process are presented in Examples 6 and 8. ##STR62## PA1 A Suzuki reaction (Miyaura et al., (1981), Synth. Comm. 11, 513; Sato et al., (1989), Chem. Lett., 1405; Watanabe et al., (1992), Synlett., 207; Takayuki et al., (1993), J. Org. Chem. 58, 2201; Frenette et al., (1994), Tet. Lett. 35(49), 9177-9180; Guiles et al., (1996), J. Org. Chem. 61, 5169-5171) can also be used to further functionalize the aryl substituent. PA1 a) Briefly, di-protected malonate VIa and 1,2-dihaloalkane VIb or cyclic sulfate VIc (synthesized according to K. Burgess and Chun-Yen KE (Synthesis, (1996), 1463-1467) are reacted under basic conditions to give the diester VId. PA1 b) A regioselective hydrolysis of the less hindered ester is performed to give the acid VIe. PA1 c) This acid Vle is subjected to a Curtius rearrangement to give a racemic mixture of 1-aminocyclopropylcarboxylic acid derivatives VIf with R.sup.1 being syn to the carboxyl group. A specific embodiment for this synthesis is presented in Example 9. PA1 d, e) Alternatively, selective ester formation from the acid VIe with an appropriate halide (P*Cl) or alcohol (P*OH) forms diester VIg in which the P* ester is compatible with the selective hydrolysis of the P ester. Hydrolysis of P ester provides acid VIh. PA1 f) A Curtius rearrangement on VIh gives a racemic mixture of 1-aminocyclopropylcarboxylic acid derivatives Vli with R.sup.1 group being anti to the carboxyl group. A specific embodiment for this synthesis is presented in Example 14. PA1 1) enzymatic separation (Examples 13, 17 and 20); PA1 2) crystallization with a chiral acid (Example 18); or PA1 3) chemical derivatization (Example 10). PA1 a) A protected glycine ester derivative such as imine IXa is alkylated with an homoallylic electrophile IXb using an appropriate base such as a metal hydride, hydroxide or alkoxide. Useful leaving groups in IXb include halogens (X=Cl, Br, I) or sulfonate esters (mesylate, tosylate or triflate). The allylic alcohol functionality in IXb is protected with hydroxyl protecting groups well known in the art (e.g. acetate, silyl, acetals). PA1 b) In a second step, the hydroxyl function of monoalkylated derivative IXc is de-protected and converted to a suitable electrophilic function X such as described above for compound IXb. PA1 c) Cyclization of IXd to cyclobutane derivative IXe is carried out by treatment with a base (metal hydrides, alkoxides), followed by hydrolysis using aqueous mineral acids and neutralization with a mild base. At this stage, syn and anti-isomers of IXe can be separated by flash chromatography. PA1 d) Optionally, the double bond in IXe can also be hydrogenated under standard conditions to yield the corresponding saturated derivative IXf. PA1 coupling a peptide selected from the group consisting of: APG-P3-P2; or APG-P2; PA1 with a P1 intermediate of formula: ##STR67## PA1 coupling a (suitably protected) amino acid, peptide or peptide fragment with a P1 intermediate of formula: ##STR68## PA1 coupling a (suitably protected) amino acid, peptide or peptide fragment with an intermediate of formula: ##STR69## PA1 wherein R.sup.1 is C.sub.1-6 alkyl, cycloalkyl or C.sub.2-6 alkenyl, all optionally substituted with halogen, for the preparation of: 1) a serine protease inhibitor peptide analog, or 2) a HCV NS3 protease inhibitor peptide analog. PA1 wherein CPG is a carboxyl protecting group, for the preparation of: 1) a protease inhibitor peptide analog, or 2) a serine protease inhibitor peptide analog. PA1 wherein R.sup.1 is C.sub.1-6 alkyl, cycloalkyl or C.sub.2-6 alkenyl, all optionally substituted with halogen, for the preparation of a compound of formula I as defined above. PA1 wherein R.sub.21A is C.sub.1-6 alkyl; C.sub.1-6 alkoxy; lower thioalkyl; halo; amino optionally mono-substituted with C.sub.1-6 alkyl; C.sub.6, C.sub.10 aryl, C.sub.7-16 aralkyl or Het, said aryl, aralkyl or Het optionally substituted with R.sub.22 wherein R.sub.22 is C.sub.1-6 alkyl, C.sub.1-6 alkoxy, amido, (lower alkyl)amide, amino optionally mono- or di-substituted with C.sub.1-6 alkyl, or Het, and R.sub.21B is C.sub.1-6 alkyl, C.sub.1-6 alkoxy, amino, di(lower alkyl)amino, (lower alkyl)amide, NO.sub.2, OH, halo, trifluoromethyl, or carboxyl;
Several studies have reported compounds inhibitory to other serine proteases, such as human leukocyte elastase. One family of these compounds is reported in WO 95/33764 (Hoechst Marion Roussel, 1995). The peptides disclosed in this application are morpholinylcarbonyl-benzoyl-peptide analogues that are structurally different from the peptides of the present invention.
One advantage of the present invention is that it provides tripeptides that are inhibitory to the NS3 protease of the hepatitis C virus.
A further advantage of one aspect of the present invention resides in the fact that these peptides specifically inhibit the NS3 protease and do not show significant inhibitory activity at concentrations up to 300 .mu.M against other serine proteases such as human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), or bovine pancreatic chymotypsin, or cysteine proteases such as human liver cathepsin B (Cat B).
A further advantage of the present invention is that it provides small peptides of low molecular weight that may be capable of penetrating cell membranes and may be active in cell culture and in vivo with good pharmacokinetic profile.